The mitotic regulator polo‐like kinase 1 as a potential therapeutic target for c‐Myc‐overexpressing canine osteosarcomas

Abstract Osteosarcoma is the most common primary malignant bone tumour in dogs, characterized by a locally aggressive and highly metastatic behaviour. Despite the current standards of care, most dogs succumb to the disease, indicating the need for novel treatment strategies. Polo‐like kinase 1 (PLK1) is dysregulated in a variety of human cancer types, including osteosarcoma, and induces c‐Myc accumulation. The crosstalk between the two molecules coordinates cell proliferation, differentiation, self‐renewal and apoptosis. Therefore, PLK1 has recently emerged as a potential therapeutic target, mainly in tumours overexpressing c‐Myc. BI 2536 is a selective PLK1 inhibitor promoting mitotic arrest and apoptosis in a variety of cancer cells. This research aimed at evaluating PLK1 and c‐Myc protein expression in 53 appendicular canine osteosarcoma (cOSA) samples and the in vitro effects of BI 2536 on a c‐Myc and PLK1‐overexpressing cOSA cell line (D17). PLK1 and c‐Myc expression in cOSA samples showed no correlation with clinicopathological data. However, c‐Myc overexpression was associated with a significantly reduced overall survival (p = .003). Western Blot and RT‐qPCR assays revealed that D17 expressed high protein and transcript levels of both PLK1 and MYC. When treated with BI 2536 (range 2.5–15 nM) for 24 h, D17 showed a substantial decrease in cell growth, inducing apoptosis and G2/M cell cycle arrest. Interestingly, under BI 2536 treatment, D17 showed decreased c‐Myc protein levels. Consistent with human OSA, these preliminary data outline the prognostic value of c‐Myc expression in cOSA and highlight the potential role of PLK1 as an antiproliferative therapeutic target for tumours overexpressing c‐Myc.


| INTRODUCTION
Osteosarcoma (OSA) represents the most common primary bone tumour in dogs [1][2][3] and is characterized by a locally aggressive and highly metastatic behaviour. 1 Despite the current standard of care, most dogs succumb to the disease within a year from the diagnosis. 1 In addition, canine OSA (cOSA) shares several clinical, histopathological and molecular features with the human counterpart, including aberrant expression and mutations of driver genes, 2,3 hence representing an excellent model in comparative oncology. 1 Polo-like kinase 1 (PLK1) is a serine/threonine kinase 4 playing a crucial role in cell cycle regulation and mitotic process [5][6][7] by acting on chromosome segregation, spindle assembly and cytokinesis. 8 Studies have demonstrated that PLK1 is usually overexpressed in a variety of cancers in human, including OSA (hOSA), and is broadly associated with a poor prognosis and disease progression. [9][10][11] Conversely, its role in dogs remains unclear.
c-Myc is a vital transcriptional regulator involved in cell cycle control, apoptosis, and protein synthesis. Additionally, MYC is one of the most commonly activated oncogenes in human and canine tumours being associated with tumourigenesis and sustained tumour growth. [12][13][14] In hOSA, c-Myc is frequently overexpressed and correlated with the development of metastases and a poor prognosis. 12,[15][16][17] Although aberrant activation of c-Myc pathway genes has been previously reported, 3,13 only recently MYC activation was correlated with a short disease-free interval in cOSA. 14 It is within this context that functional studies demonstrated that PLK1/Fbw7/c-Myc axis creates a positive auto-regulatory signal, sustaining the mutual increased expression of these genes. 18,19 In particular, PLK1 plays a key role in c-Myc protein stabilization and accumulation in the cytoplasm, allowing its migration into the nucleus where it promotes G 2 /M transition and acting as a transcriptional factor.
These findings underline the importance of PLK1 inhibitors as promising selective therapies against c-Myc-overexpressing canine tumours, as previously demonstrated by the administration of Volasertib 20,21 in a number of human cancer subtypes.
In a recent RNA-seq study, both PLK1 and MYC oncogenes were found overexpressed in two well-established cOSA cell lines, suggesting a potential implication of this signalling axis in this tumour. 3 BI 2536, was the first selective PLK1 inhibitor able to promote mitotic arrest and apoptosis in MG-63 human OSA cell line 22 and xenografts models. 23 Although Volasertib was developed from BI2536 given the more favourable pharmacokinetic properties, 20 both molecules are potent PLK1 inhibitors with superimposable in vitro effects, including inhibition of c-Myc expression and consequent cell death. 18 These findings prompted us to investigate the role of PLK1 and c-Myc in cOSA and to evaluate the in vitro biological effects of BI 2536 treatment 24 on a PLK1 and c-Mycoverexpressing cOSA cell line.

| Histological diagnosis and immunohistochemistry
Tissues were placed in an EDTA-based decalcification solution (Bio-Optica, Milano, IT) until sufficient demineralization, before processing for histopathology. Formalin-fixed, paraffin-embedded (FFPE) tumour samples were stained with haematoxylin-eosin (HE) for diagnosis. The histological classification was performed according to the World Health Organization (WHO) guidelines 25  were used for detection. All antibodies were validated for crossreactivity with canine positive controls 27 ( Figure S1).
Immunolabelled slides were randomized and masked for blinded examination, which was performed by two independent pathologists.
Immunohistochemical evaluation of PLK1 and c-Myc expression was performed using previously reported scoring systems and detailed in Table S1. 28,29 2.3 | Cell line selection and culture conditions

| Cell cycle analysis by FACS
The biological effect of BI 2536 on the cell cycle was evaluated by

| Clinicopathological data
This retrospective study included 53 dogs with appendicular OSA.
The clinicopathological characteristics and follow-up data are pro-   Table 2, and representative images are shown in Figure 3.

| PLK-1 inhibition in vitro reduces c-Myc protein expression
To investigate the effect of PLK1 inhibition on c-Myc expression, WB and RT-qPCR were performed on D17 cells exposed to BI 2536. As depicted in Figure 8   Since c-Myc overexpression is known to predict outcome in hOSA, 12,17,47 we hypothesized that expression of the PLK1 in addition to c-Myc may replicate a similar result in cOSA. In this study, c-Myc overexpression was associated to a negative outcome in our samples, supporting his role as an emerging prognostic biomarker. 48